Chemical assays

ABSTRACT

An assay device in which to carry out a fluid-phase chemical assay, comprising means for supporting a test substrate, a sample chamber for receiving a fluid sample and at least one fluid control device for controlling the movement of fluid into and/or out of the sample chamber, wherein the fluid control device comprises a fluid outlet chamber in fluid communication with the sample chamber, and a displaceable flexible diaphragm the displacement of which alters the volume of the outlet chamber so as to allow and/or restrict fluid flow between the outlet and sample chambers. The invention also provides assay apparatus incorporating such a device, an assay station for use as part of such apparatus, a fluid control unit for use as part of the assay device and a method of conducting an assay which may involve the use of such apparatus and devices.

This application is the US national phase of international applicationPCT/GB 01/05158 filed 22 Nov. 2001, which designated the US.

FIELD OF THE INVENTION

This invention relates to methods, apparatus and devices for use incarrying out chemical (which includes biochemical) assays, in particularfor the detection of biological materials such as proteins and peptides.

BACKGROUND TO THE INVENTION

Certain types of chemical, in particular biochemical, assays involveimmobilising on a test substrate a probe species capable of bindingselectively to a target species. A fluid sample, containing or suspectedto contain the target species, is brought into contact with the testsubstrate; target species present in the sample will then bind to theimmobilised probe. After washing the substrate to remove unboundspecies, the presence of the target-probe pair can be detected inseveral known ways, including via chemical “labels” (for instance,labels capable of chemiluminescence or fluorescence) attached to thetarget species.

This principle is used in a large number of biochemical assays, forinstance to detect the presence of target nucleotide sequences orproteins. It involves, however, an often complex sequence of procedures.A suitably selective probe for the target species must firstly beidentified, usually by means of some form of screening, and immobilisedon a test substrate. A sample fluid must then be maintained in contactwith the substrate for a sufficient period of time, and under suitableconditions, for target-probe binding to take place (and to take place toa detectable degree). During this period, the temperature of the sampleoften needs to be cycled between quite precise ranges and over specifictime periods, to enable binding to occur. The test substrate must thenbe washed, usually with increasing levels of stringency to remove notonly unbound species but also those which are bound with an unacceptablylow degree of specificity. Finally, the washed substrate must beanalysed to detect the presence and/or amount of target-probe pairs.

These procedures can to an extent be automated, but often still involvesignificant manual intervention, for instance to control theintroduction of samples and reagents at appropriate times and locations.Moreover, apparatus for carrying out the procedures can be both complexand costly, involving large numbers of separate fluid control devices(valves and pumps) in order to introduce what is often a large number ofnecessary sample and/or reagent fluids.

Since it may be desirable to assay a large number of samples at a time,and/or to test a sample against a large number of probe species, thereis a constant need to enhance the efficiency of such assays, to reducethe complexity of the apparatus in which they are carried out, tominimise the amount of manual intervention needed, to maximisethroughput and/or to increase accuracy and consistency in the results.Furthermore, since the samples being assayed are often scarce (forinstance, DNA-containing samples), and typically need to be screened formore than one target species, it is always desirable to minimise theamount of sample needed for an assay, typically by increasing detectionsensitivity.

It is already known to carry out a chemical assay by spreading a thinlayer of a liquid sample over a flat test substrate, such as a glassmicroscope slide, on which an “array” of several, often hundreds ormore, probe species has been immobilised. This allows the sample to bescreened simultaneously for a corresponding number of target species.Such arrays have, for instance, been disclosed recently for thedetection of proteins in a biological sample; the test substrate may bereferred to as a “protein array” or “protein biochip” [de Wildt, R M Tet al, Nat Biotechnol, 18 (9), 989-94 (September 2000); Mendoza, G,BioTechniques, 27 (4), 781-788 (1999); Bussow, K et al, Genomic 65, 1-8(2000)]. It would be desirable to be able to use such substrates in anat least partly automated assay process, and preferably to be able toprocess a plurality of substrates simultaneously.

STATEMENTS OF THE INVENTION

According to a first aspect of the present invention there is providedan assay device in which to carry out a fluid-phase chemical assay, thedevice comprising (i) means for supporting a test substrate, (ii) asample chamber for retaining a fluid sample in contact with a testsubstrate which is so supported and (iii) at least one fluid controldevice for controlling the movement of fluid into and/or out of thesample chamber, wherein the fluid control device comprises a fluidoutlet chamber in fluid communication with the sample chamber, and adisplaceable flexible diaphragm the displacement of which alters thevolume of the fluid outlet chamber so as to cause and/or allow and/orrestrict fluid flow between the fluid outlet chamber and the samplechamber.

The term “fluid-phase chemical assay” means a diagnostic test fordetecting the presence and/or quantity of a target species in a fluidsample, by means of a chemical reaction. It is intended to embracebiochemical assays such as for the detection of a target nucleotidesequence (DNA, RNA etc.) or protein or peptide. It will typicallyinvolve the use of a probe species immobilised on a test substrate withwhich the sample is brought into contact, the probe species beingcapable of reacting with the target species, via which reaction thepresence and/or quantity of the target species may be detected. Thereaction suitably involves selective binding of the probe species to thetarget species, the bound pair being detectable using conventionaltechniques such as fluorescence, chemiluminescence, coloured dyes andthe like.

The means for supporting a test substrate may include means, such asclamps, spring clips and the like, for securing the substrate in placein the assay device. It preferably also includes sealing means, such asan appropriately shaped gasket or an O-ring seal, for sealing areas ofcontact between the test substrate and the rest of the device, inparticular to help define, and to prevent fluid leakage from, the samplechamber.

The assay device may be capable of supporting two or more testsubstrates.

The sample chamber may be defined at least partly by a test substratewhich, in use, is supported in the device. The sample chamber shouldthen be an enclosed space, save for fluid inlets and/or outlets such asthose providing fluid communication with the fluid control device(s).Preferably the sample chamber volume is small, typically between 50 and120 μl, such as between 100 and 120 μl. More preferably it can enclose athin layer of the sample fluid, for instance between 50 and 100 μm deep,adjacent the active surface of a test substrate supported in the device.The “active surface” of the substrate is that part of its surface whichcarries one or more probe species; the sample chamber ideally allows theenclosed fluid to contact the whole of the active surface. Thedimensions of the active surface are typically about 20 mm by between 30and 65 mm.

Again, the assay device may include two or more sample chambers whichmay, in use, be associated with separate test substrates or withdifferent regions of a single substrate.

The fluid control device may comprise a fluid flow control valve such asfor controlling the introduction of fluid to and/or evacuation of fluidfrom the sample chamber. It may comprise a fluid agitation device forinducing fluid movement within the sample chamber, such as by forcingfluid into and/or out of the sample chamber. It may comprise a fluidstorage device in which a quantity of fluid may be held prior to itsintroduction into or following its evacuation from the sample chamber oranother part of the assay device. (The word “comprise” is used in thisspecification to mean either “be” or “include”.)

The fluid control device is preferably itself controllable using acontrol fluid, which may be supplied to a region of the flexiblediaphragm such that variations in the control fluid pressure causedisplacement (which term includes distortion) of the diaphragm. Thefluid control device therefore preferably comprises a control chamberand a control port through which control fluid may be introduced intothe control chamber, the diaphragm being arranged between the controlchamber and the fluid outlet chamber in such a way that displacement ofthe diaphragm, caused by pressure changes in control fluid supplied tothe control chamber, alters the volume of the outlet chamber. A suitablecontrol fluid is compressed air, although many other pressurised liquidsor gases could be useable to the same effect.

Instead or in addition, the fluid control device may be at leastpartially controlled by varying the pressure of one or more of the otherfluids (eg, sample or reagent fluids) supplied to it.

The flexible diaphragm should be made from, at least at its surface, amaterial which is inert with respect to the reagents which will passthrough the assay device in use. It must have sufficient resilience tofunction in the required manner under the fluid pressures likely to beapplied to it, ie, to be displaceable and/or distortable between therequired operating positions. Suitable diaphragm materials includesilicone rubbers of hardness 40 to 60 Shore A and thickness between 0.3and 2 mm, typically 1 mm. These may optionally be faced with low or highdensity polyethylene (LDPE or HDPE) or polypropylene, of a filmthickness between 10 and 100 μm.

Where the fluid control device comprises a valve, it preferablyadditionally comprises a fluid inlet chamber, the flexible diaphragmbeing displaceable between a first position in which it restricts orprevents fluid communication between the fluid inlet and outlet chambersand a second position in which fluid communication between the inlet andoutlet chambers is allowed.

The inlet and outlet “chambers” may take the form of fluid conduits.Communication between them may be directly or via one or moreintermediate chambers and/or conduits. In general in this specification,the term “fluid communication” embraces both direct and indirectcommunication, though preferably direct unless otherwise specified.

Where the fluid control device comprises a fluid agitation device, againthe diaphragm is preferably arranged between a control chamber and thefluid outlet chamber in such a way that displacement of the diaphragm,caused by pressure changes in control fluid supplied to the controlchamber, alters the volume of the outlet chamber. In this way,displacement of the diaphragm can cause fluid to be forced either intoor out of the sample chamber, thus generating fluid movement within thesample chamber. Such movement is generally desirable to maintain ahomogeneous sample fluid and hence increase accuracy and sensitivity ofan assay.

Preferably, the assay device of the invention incorporates two suchfluid agitation devices, which can be reciprocally operated to movefluid back and forth through the sample chamber. In such an arrangement,the two agitation devices preferably communicate with opposite ends ofthe sample chamber, or at least with two spaced apart regions of thesample chamber.

Where the fluid control device comprises a fluid storage device, itpreferably comprises a fluid inlet port for receiving fluid (typically asample fluid) and a fluid storage chamber, in fluid communication withthe inlet port, for holding fluid received at the inlet port. Thediaphragm then preferably functions to control movement of fluid intoand out of the storage chamber, being displaceable between a firstposition in which fluid is held in the storage chamber, and a secondposition in which fluid is urged out of the storage chamber and into theoutlet chamber. Control of the diaphragm, to displace it between thesefirst and second positions, may be effected by means of an associatedvalve and/or by the application of a pressure change to another part ofthe control device, for example directly to the storage chamber, moreparticularly by the supply of control fluid to the diaphragm to displaceit within the storage chamber.

Communication between the storage and outlet chambers may be via anintermediate chamber. Moreover, a single port may function as both inletand outlet, the relative fluid pressures (i) in the storage and/orintermediate chambers and (ii) at the inlet/outlet port determining thedirection of fluid flow, and the diaphragm position either allowing orpreventing flow as desired. This arrangement may effectively comprise acombination of a fluid storage device and a diaphragm-operated valve tocontrol the introduction of fluid into it, for example via theintermediate chamber.

The storage chamber typically holds a small volume, for instance between50 and 200 μl, preferably between 100 and 150 μl, of fluid. It ideallyholds at least enough fluid to fill the associated sample chamber; a 90μl sample chamber may for instance be associated with a 150 μl storagechamber, which can of course be part filled if appropriate. The storagechamber is particularly suited for the storage of small quantities ofscarce sample fluids, which may be pre-loaded into the assay device andstored in close proximity to the sample chamber, for introduction intothe sample chamber at an appropriate point in an assay.

Where the fluid control device has a fluid inlet port or inlet conduit,the port or conduit may be of any size and shape suitable to allow theintroduction of fluid for example from a source elsewhere in the assaydevice or, in the case of a sample fluid, conveniently via a needle orpipette. The inlet port or conduit may for example have an opening tothe exterior of the device, the opening being adapted to receive apipette or other fluid introducing means.

The fluid control device may comprise a fluid loading device, into whichfluids may be loaded and/or evacuated and/or transferred either fromoutside the assay device or from other component(s) within the assaydevice. Such a fluid loading device comprises a receptacle, such as acup- or bowl-shaped receptacle, to accommodate fluid which may beintroduced into it for instance from an external source. Preferably, thereceptacle is directly accessible from the outside of the assay device,and most preferably, it is adapted to receive a fluid introducing meanssuch as a pipette.

A cup- or bowl-shaped receptacle may conveniently be provided in theexterior surface of a plate or block forming part of the assay device,as described above. Its capacity may suitably be between 10 and 500 μl,preferably between 50 and 100 μl, depending on its intended use. It willhave at least a first outlet which provides fluid communication withanother part of the assay device, typically a fluid storage device orthe sample chamber, such communication conveniently being via anotherfluid control device such as a valve.

The receptacle preferably also has a second outlet through which fluidmay be evacuated, typically to waste. The locations of the first andsecond outlets will depend on their intended functions; suitably thesecond is positioned, in use, at a higher fluid level within thereceptacle than the first.

Preferably at least the first outlet is in direct fluid communicationwith a valve for controlling fluid flow into and/or out of the fluidloading device. More preferably still, the first outlet is in directfluid communication with, or constitutes, the fluid inlet chamber of avalve of the type described above which is operated via a displaceableflexible diaphragm.

Thus, in a fluid control device (in particular a valve) which forms partof an assay device according to the invention, any fluid inlet chamberor port preferably is or comprises a fluid loading device of the typedescribed above.

The assay device of the invention preferably comprises more than one,typically two or more, for instance two, such fluid loading devices,which may then be used for loading fluids from externally and/or fortransferring fluids between other components of the device (eg, samplechambers, fluid storage devices and reagent or other fluid sources).Each loading device may be associated with (ie, in direct or indirectfluid communication with) one or more storage devices and/or samplechambers, and/or with one or more other fluid loading devices so thatfluid may be transferred between the loading devices for example via acommonly connected storage device. The capacity of each loading device(ie, of its fluid receptacle) is ideally larger than that of anassociated sample chamber and/or storage device, by an amount sufficientto accommodate losses and “dead” volumes and still to provide sufficientfluid to fill the relevant chamber/device. Its capacity may for examplebe between 10 and 100% greater than that of the associatedchamber/device.

The assay device of the invention preferably comprises more than onefluid control device of the types described above. It may for instancecomprise both a fluid inlet and a fluid outlet valve, controllingrespectively the introduction of fluid into and evacuation of fluid fromthe sample chamber. It may comprise more than one fluid inlet valve,allowing the introduction of more than one fluid into the samplechamber. Fluid inlet valves may also be provided for controlling theintroduction of fluid(s) into one or more fluid storage devices. Theassay device may comprise one or more fluid receiving ports associatedwith one or more of the inlet valves. It preferably additionallycomprises one or more fluid agitation devices, preferably at least two.More preferably it additionally comprises one or more fluid storagedevices, in which fluid may be held in close proximity to the samplechamber. Not all of the fluid control devices need be in direct fluidcommunication with the sample chamber.

Ideally the assay device comprises at least three, more preferably atleast four or five or six fluid control devices, the fluid ports andchambers of which are defined within a single unit which may also serveat least partly to support a test substrate. The assay device can thuscomprise integral fluid control devices, which may be supplied fromexternally with appropriate sample, reagent, wash and control fluids.This allows the device to be relatively simple and compact inconstruction. It also facilitates independent temperature control, flowrate analysis and other necessary processes for a test substratesupported within the device.

The unit in which the fluid control devices are provided may comprise,for instance, a block or plate made from a suitable material, such as ametal or plastics material, in which the necessary fluid chambers,conduits and ports may be machined, moulded or similarly provided.Chambers, conduits and ports may be provided at the face of such a blockor plate and may be at least partly defined by a sealing layer, such asa gasket, positioned adjacent that face.

More preferably still, the assay device of the invention comprises asingle flexible diaphragm common to more than one, ideally all, of itsfluid control devices. In other words, a single diaphragm is arranged toperform more than one function at different locations in the assaydevice, the locations corresponding to the relevant fluid chambers inthe fluid control devices. Typically a separate means for controllingthe device operation will be needed at each such location; this maycomprise a separate control chamber or port, for bringing a controlfluid into contact with the diaphragm at the relevant location to causea local displacement of the diaphragm. All the control ports may, inuse, be supplied with control fluid from a single source, optionallywith a separate valve or other means to control the supply of fluid toeach control port. These features can again simplify the construction ofthe assay device.

The diaphragm may for example be positioned between two adjacent platesor blocks, each providing certain fluid chambers and channels to formpart of the fluid control devices. In a more preferred embodiment, theassay device comprises more than two (for instance three) stackedplates, with a diaphragm positioned between each pair of adjacentplates, so that different fluid control devices can be defined bydifferent plate pairs. This increases the versatility of the system,allowing a wider range of devices to be provided in a single unit and ina wider range of locations within that unit.

One or more of the plates may also serve as a support for a testsubstrate.

In regions of the device where the diaphragm contacts, and provides aseal around, the edges of a fluid conduit or chamber defined in anadjacent plate or block, raised surface elements (eg, ridges) or otherforms of surface profiling may be provided adjacent or close to theperimeter of the conduit or chamber, so as to amplify, in the region ofthe conduit or chamber perimeter, the force applied to clamp thediaphragm in position adjacent the plate or block. Such surfaceprofiling may be provided on the surface(s) of either or both of theplates between which the diaphragm is clamped (preferably that in whichthe relevant conduit or chamber is defined), and/or on the diaphragmitself.

Ideally the assay device of the invention also incorporates a fluiddistribution assembly, by means of which the necessary fluid(s) may beintroduced into the device from external sources and subsequentlyremoved from the device. This assembly will typically include one ormore fluid inlet ports, directly or indirectly connectable to externalsources of for instance reagent and wash fluids, and one or moreconduits through which fluid may pass from the inlet port(s) to thefluid control device(s) of the assay device. It will also include one ormore fluid outlet ports, directly or indirectly connectable for instanceto a waste reservoir, and one or more conduits through which fluid maypass from the assay device to the outlet port(s).

Again, the necessary fluid channels may be drilled, extruded, machinedor moulded within a unit such as a plate or block which is appropriatelypositioned with respect to the fluid control device(s) of the assaydevice. Chambers, conduits and ports may be provided at the face of sucha plate or block and may be at least partly defined by a sealing layer,such as a gasket, positioned adjacent that face. Most preferably, thefluid ports and conduits of the distribution assembly are provided inthe same plate or block in which the fluid control devices, or partsthereof, are located, or at least in an adjacent plate or block.

The inlet ports of the distribution assembly may in certain casescorrespond to those of the fluid control devices and may for examplecomprise fluid loading devices of the type described above. One or moreof the inlet ports may be for the introduction of a control fluid suchas compressed air.

The incorporation of such a fluid distribution assembly allows for aplurality (often a very large number) of assay devices to be connectedto a common set of fluid supply and removal lines, and to the controlsfor such fluid lines, and hence to be simultaneously processed in asingle assay apparatus.

A second aspect of the present invention provides an assay device inaccordance with the first aspect, in combination with a test substrateon which one or more probe species are immobilised. The substrate may,for example, be a glass slide. The probe species may be immobilised onthe substrate in any known manner. Preferably the substrate carries aplurality (for instance, up to about 100,000, typically between about5,000 and 20,000) of immobilised probe species, in any suitablearrangement such as in an array.

According to a third aspect of the present invention, there is provideda device for monitoring the flow rate of a first fluid, typically aliquid, the device comprising a primary measuring chamber through whichthe first fluid may flow, a fluid inlet port upstream of the primarymeasuring chamber, through which a volume of a second fluid (typically agas, such as in the form of a bubble) may be introduced into the firstfluid flow, and primary detection means, associated with the primarymeasuring chamber, for detecting the presence of the second fluid in thefirst fluid as they pass through the primary measuring chamber. Thedetection means may be electrical in operation, detecting changes forexample in conductance or capacitance between electrical contactspositioned at different locations in the flow path of the first fluidthrough the measuring chamber. A printed circuit board may for instancebe provided in, and conveniently form one wall of, the measuringchamber, to detect the presence or absence of the second fluid in themeasuring chamber—where the first fluid is a liquid and the second aninjected gas bubble, for instance, absence of the liquid indicates thepresence of the gas bubble, and can be detected using the printedcircuit board.

Alternatively, optical detection means may be used, such as aredescribed for instance in U.S. Pat. No. 4,210,809.

The time taken for the second fluid to reach the primary measuringchamber, from its inlet port, may thus be measured and used to providean indication of the flow rate of the first fluid through the device.

Preferably, the monitoring device additionally comprises a secondarymeasuring chamber, in fluid communication with and convenientlydownstream of the primary one, the secondary chamber having associatedwith it a secondary detection means, for detecting the presence of thesecond fluid in the first as they pass through the secondary measuringchamber. A more accurate indication of the first fluid flow rate maythen be obtained by measuring the time taken for the second fluid totravel between the two measuring chambers. Fluid communication betweenthe measuring chambers is preferably by means of an extended, morepreferably labyrinthine, fluid conduit, to increase the distancetravelled by the first and second fluids between the measuring chambers.

The flow rate monitoring device of this third aspect of the inventionmay be used in association with an assay device according to the firstaspect, to measure the rate of flow of one or more fluids through theassay device. The monitoring device is preferably incorporated in, morepreferably integral with, the assay device, conveniently downstream ofthe sample chamber. This can be achieved, for instance, by providing themeasuring chamber(s), fluid conduit(s) and inlet port(s) of themonitoring device in a unit containing the fluid control device(s).

An assay device according to the first aspect of the inventionpreferably incorporates means for controlling the temperature inside thesample chamber; this may be particularly useful in the case where abiochemical assay involving thermal cycling is to be carried out in thedevice. The temperature control means may include conventional devicessuch as hot air blowers, ovens, fans, fluid heating and/or coolingbaths, etc. The assay device may for instance comprise a heat sink, ofconventional construction, which can be cooled for instance by means ofa fan which forces a cooling fluid (such as air) through channelsprovided in it, and which can preferably also be heated for instanceelectrically. Heat may then flow by conduction between the heat sink andthe rest of the device, at least in the region of the sample chamber.

Instead or in addition, the temperature control means may comprisechannels within the device or its surrounding apparatus, through which aheating/cooling fluid may be caused to flow. This fluid may beexternally heated and/or cooled by any convenient means, for instanceelectrical resistance heaters, forced (or natural) air-cooled heatexchangers or peltier devices. It may be circulated by convection or,preferably, by means of a pump. Such a form of temperature control cangive improved temperature uniformity, both across the test substrate andalso between assay devices where several are to be processed together.It may make possible more rapid temperature changes, in particular ifseveral external reservoirs of heating/cooling fluids are held atdifferent desired temperatures to be supplied to the assay device atappropriate times. In use, several assay devices may be supplied by acommon source or sources of heating/cooling fluid(s).

For more efficient heating and/or cooling the assay device, or ifappropriate groups of assay devices, is/are preferably enclosed in achamber to isolate it/them from neighbouring assay devices and from thesurrounding environment.

According to a fourth aspect of the present invention, there is providedapparatus for carrying out a fluid-phase chemical assay, the apparatuscomprising an assay device in accordance with the first aspect of theinvention, and/or an assay device/test substrate combination inaccordance with the second aspect, and/or a flow rate monitoring devicein accordance with the third aspect.

Such apparatus preferably comprises a plurality of assay devices inaccordance with the invention. It may comprise one or more assay“stations”, each of which can accommodate a plurality of assay devices.Each station ideally has an associated fluid distribution assembly,communicating with those of its assay devices, to enable appropriatefluids to be supplied to the assay devices and spent fluids to beremoved to waste.

A typical such assay station might be capable of supporting for instanceat least four or six or ten or twelve or sixteen assay devices.Apparatus according to the fourth aspect of the invention could includefor example at least three or four or five or ten assay stations. Thiswould allow the simultaneous execution of a large number of assays, eachin a respective assay device, and would lend itself particularly well toat least partial automation, for instance under the control of amicroprocessor. Ideally the fluid movement through each assay deviceand/or assay station could be independently controlled. Similarly, theoperating temperature could be independently controlled for at leasteach individual assay station.

A fifth aspect of the invention provides a fluid distribution system foruse in the apparatus of the fourth aspect, the system comprising firstand second fluid inlet lines via which first and second fluids may bedrawn from respective sources, first and second fluid flow controldevices, each allowing a variable fluid flow rate, in the first andsecond fluid inlet lines respectively, and control means for controllingindividually the flow rates through the first and second fluid flowcontrol devices. The system preferably additionally comprises a fluidmixing device, downstream of the fluid flow control devices, forcombining the first and second fluids emerging from the control devices.The combined fluid stream emerging from the fluid mixing device may thenbe directed to a desired location, preferably to one or more assaydevices or assay stations according to the invention.

The fluid flow control devices are preferably variable rate pumps, oralternatively valves providing adjustable flow rates.

Such a fluid distribution system allows two fluids to be combined in adesired ratio. Ideally the fluid flow rates through the first and secondfluid flow control devices are continuously variable between theirminimum and maximum values, allowing for continuous variation of thefirst and second fluid ratio in the mixture emerging from the system.This could be of particular use, for instance, in supplying varyingconcentrations of reagent or wash solutions to an assay device (in whichcase the first fluid might be a suitable reagent in concentrated formand the second fluid a diluent such as water).

The fluid distribution system may include more than two fluid inletlines with more than two respective fluid flow control devices. In thiscase any desired number of fluid mixing devices may be included toachieve any desired combination of the fluids passing through thesystem.

The fluid distribution system may form part of apparatus according tothe fourth aspect of the invention, and may be used to supply one ormore sample, reagent or wash fluids to the assay device(s). Preferablyapparatus according to the fourth aspect includes more than one suchdistribution system, for introducing more than one fluid mixture intothe assay device(s).

According to a sixth aspect of the present invention, there is provideda fluid control unit for use as part of an assay device according to thefirst aspect, the unit comprising a fluid control device as describedabove, ie, comprising a fluid outlet chamber which is connectable to anassay device sample chamber in use, and a displaceable flexiblediaphragm the displacement of which alters the volume of the fluidoutlet chamber so as to cause and/or allow and/or restrict, in use,fluid flow between the fluid outlet chamber and the sample chamber. Theunit preferably comprises a plurality of such fluid control devices,which may include (as above) valve(s), fluid agitation device(s), fluidstorage device(s) and/or fluid loading device(s).

The unit is preferably constructed from two or more adjacent plateshaving a flexible diaphragm positioned between each pair of adjacentplates, at least some of the fluid chambers and ports of the fluidcontrol device(s) being defined in those faces of the plates which areadjacent the diaphragm(s). One of the plates may also serve as a supportfor a test substrate, in use. The unit preferably also comprises means(such as fluid inlet and outlet ports) for connecting it to externalfluid inlet and outlet conduits (for instance, leading to fluid sourcesand/or to waste), to one or more supplies of a control fluid such ascompressed air, and/or to a sample chamber when the unit forms part of acomplete assay device.

The unit of the sixth aspect of the invention preferably additionallycomprises a flow rate monitoring device in accordance with the thirdaspect, and/or temperature control means as described in connection withthe first aspect.

A seventh aspect of the invention provides an assay station comprisingmeans for accommodating one or more assay devices, preferably accordingto the first aspect of the invention, and a fluid distribution assembly(for instance as described above) which is connectable, for example viafluid ports, to external fluid conduits for the supply of fluids to,and/or their removal from, assay devices held at the station, the fluiddistribution assembly having one or more fluid conduits to allow fluidcommunication between the assay devices and one or more external fluidconduits. More preferably, the assay station comprises one or more,preferably a plurality of, fluid control units in accordance with thesixth aspect of the invention, the fluid distribution assembly allowingcommunication between the external fluid conduits and each of the fluidcontrol units. More preferably still, the assay station comprises two ormore adjacent plates with a flexible diaphragm positioned between eachpair of adjacent plates, wherein the fluid control devices of theindividual units, and at least in part the fluid conduits of thedistribution assembly, are provided within the two plates.

The devices, apparatus, assemblies and units of the invention mayinclude at least partially automated control means, for instancecomprising a computer.

According to an eighth aspect of the present invention, there isprovided a method of conducting a fluid-phase chemical assay whichinvolves the operation of an assay device or other apparatus inaccordance with the invention. In particular, the method involvessupporting a test substrate in an assay device in accordance with thefirst aspect of the invention, and controlling the movement of fluidinto and/or out of the sample chamber in contact with the test substrateusing at least one fluid control device which comprises a fluid outletchamber in fluid communication with the sample chamber, and adisplaceable flexible diaphragm the displacement of which alters thevolume of the fluid outlet chamber so as to cause and/or allow and/orrestrict fluid flow between the fluid outlet chamber and the samplechamber.

The method preferably involves locating a plurality of test substratesin a corresponding number of assay devices (suitably using apparatusaccording to the fourth aspect of the invention) and conducting afluid-phase chemical assay in each device, simultaneously orsequentially.

BRIEF DESCRIPTION OF THE DRAWINGS

In the accompanying illustrative drawings:

FIG. 1 shows apparatus according to the fourth aspect of the invention;

FIG. 2 shows one of the assay stations A to D from the FIG. 1 apparatus;

FIG. 3 shows the fluid control devices for one of the cassettes seen inFIG. 2;

FIGS. 4 a and 4 b are a stylised “plan” view and a partially explodedcross section respectively of one of the valves seen in FIG. 3;

FIGS. 5 a and 5 b are a stylised plan view and a cross sectionrespectively of an alternative valve useable in the FIG. 3 cassette;

FIG. 6 is a cross section through one of the fluid agitation devicesseen in FIG. 3;

FIGS. 7 a and 7 b are a stylised plan view and a cross sectionrespectively of a combination of fluid control devices such as thoseshown in FIGS. 4 to 6;

FIGS. 8 a and 8 b are a stylised plan view and a cross sectionrespectively of an alternative combination of fluid control devices;

FIGS. 9 a and 9 b are a stylised plan view and a perspective viewrespectively of a fluid control unit in accordance with the sixth aspectof the invention, for use in the apparatus of FIGS. 1 and 2;

FIG. 10 is a section through one of the valves seen in FIG. 9 a;

FIGS. 11 a and 11 b are a stylised plan view and a cross sectionrespectively of a sample chamber of one of the cassettes seen in FIG. 2;

FIG. 12 is a cross section through an assay device in accordance withthe first aspect of the invention, for use in the apparatus of FIG. 1;

FIGS. 13 a and 13 b are a stylised plan view and a cross sectionrespectively of part of an assay station for use in the FIG. 1apparatus;

FIG. 14 is a cross section through a flow rate monitoring device inaccordance with the invention, for use in the FIG. 1 apparatus;

FIG. 15 is a cross section through part of an assay station of the typeshown in FIG. 13, in combination with temperature control means;

FIG. 16 a is a side view and FIG. 16 b a stylised plan view of a“blanked-off” cassette from the FIG. 13 assay station;

FIGS. 17 a and 17 b are a stylised plan view and a cross sectionrespectively of an alternative type of valve for use in apparatus orassay devices according to the invention;

FIG. 18 is a cross section through one embodiment of the FIG. 17 valve;

FIG. 19 is a cross section through an alternative embodiment of the FIG.17 valve;

FIG. 20 is a cross section through a fluid loading device for use in anassay device according to the invention;

FIG. 21 is a section through part of the FIG. 20 device during a typicalfluid loading operation;

FIG. 22 shows an arrangement of fluid loading devices and other fluidcontrol devices useable in an assay device according to the invention;

FIGS. 23 a, b and c are sections through parts of fluid control devicesin accordance with the invention, illustrating an alternativeconstruction;

FIGS. 24 a and b are respectively a section through part of a fluidcontrol device in accordance with the invention and a stylised plan viewof the same;

FIG. 24 c is a section corresponding to that in FIG. 24 b showing thedevice during operation;

FIG. 25 is a section corresponding to that in FIG. 24 a but through partof an alternative fluid control device in accordance with the invention;and

FIGS. 26 a and 26 b are a stylised plan view and a cross sectionrespectively of an alternative type of valve for use in apparatus orassay devices according to the invention.

All figures are schematic.

DETAILED DESCRIPTION

Embodiments of the present invention will now be described, by way ofexample only, with reference to the accompanying drawings.

FIG. 1 shows schematically apparatus according to the fourth aspect ofthe invention, for use in carrying out several simultaneous chemicalassays, in particular using protein arrays. The apparatus comprisesreservoirs 1 of the required reagent fluids (including buffers,detergents, catalysts, wash solutions and the like and typically also,for in situ dilution of other reagents, distilled water and/or othersolvents). An appropriate number of fluid supply lines, here illustratedschematically as a single conduit 2, carries fluids from the reservoirs1 to assay stations 3, each of which houses a number of slide“cassettes” as described below in connection with FIGS. 2 and 3. Theassay stations are here labelled A to D; in apparatus according to theinvention there can be any desired number of such stations supplied fromthe same fluid reservoirs. The apparatus of the invention allows allstations to be processed simultaneously but also, if necessary,independently.

Supply line 4 carries a control fluid such as compressed air, via pump5, to each of the assay stations 3. Conduits 6 carry fluids from theassay stations to a waste reservoir 7.

FIG. 2 shows, also schematically, one of the assay stations A to D fromthe FIG. 1 apparatus. The station supports a suitable number of, in thiscase twelve, slide “cassettes” 8. Each cassette holds one testsubstrate, typically a microscope slide having an array of probematerials (for example, antigens and/or antibodies) immobilised on it.Each cassette also provides, in association with the assay station, thefluid control devices necessary to conduct fluids to and from the testsubstrate it holds; these devices are described below in connection withFIGS. 3 to 10 and 17 to 25.

The assay station also includes fluid conduits, valves and pumps tocontrol the flow of reagent and control fluids into and out of the slidecassettes. Fluid supply line 4 carries the control fluid (in this casecompressed air) to the slide cassettes, via a corresponding number ofvalves 9. Reagent fluids are fed to the cassettes via fluid supply lines10 (corresponding to conduit 2 in FIG. 1), selecting valves 11, pumps 12and a further mixer 13. Any desired number of valves 11, pumps 12 andmixers 13 may in practice be used, according to requirements. A “purgevalve” 14 allows the fluid conduits in the station to be purged to waste(reservoir 7, as shown in FIG. 1) with a chosen reagent fluid, bypassingthe slide cassettes 8. Fluids pumped through the cassettes drain to asingle outlet conduit 15 (corresponding to 6 in FIG. 1) and thence tothe waste reservoir.

The FIG. 2 apparatus incorporates a fluid distribution system inaccordance with the fifth aspect of the invention, which allows therelative concentrations of certain reagent fluids to be automaticallyand continuously varied.

Each valve 11 can in this case be selected to feed one of a pair offluids, for instance either that carried by conduit 10 a or that carriedby conduit 10 b, into a pump 12. The output of two or more (in this caseall three) of the pumps 12 is combined in the mixing device 13, and theresultant mixture can then be fed to the slide cassettes. If only one ofthe pumps 12 is operated, then only a single fluid is sent to thecassettes. If more than one of the pumps is operated then a mixture offluids may be supplied. By varying the pumping rate for each pump thenthe overall volumetric flow rate may be set as well as the mixingratio(s) between the fluids feeding the pumps. In this way, either apreset mix ratio may be achieved or, if the pumps are operated at acontrolled, time-varying rate, the constitution of the fluid mixture canbe varied with time. For instance, two pumps may be operated with aconstant combined pumping rate, but varying their pumping rate ratiobetween 1:0 and 0:1. The composition of the resulting fluid mixture maythereby be varied from 100% of one fluid to 100% of another, thevariation following any desired pattern with time, whether continuous orstep-wise.

This arrangement is of particular use for instance in supplying washsolutions of varying concentrations to the test substrates. Aconcentrated wash solution may be supplied to one of the pumps 12 and adiluent such as distilled water to another, allowing the wash solutionto be diluted to any desired level by altering the two pump rates.

When there are three pumps, as shown in FIG. 2, two of them may be usedto pump concentrated active ingredients, with variable blending betweenthe two, whilst the third pumps a diluent (again typically water) to setthe overall concentration of the active ingredient mixture. In this waythe FIG. 1 apparatus need only be fed from smaller bottles ofconcentrated active ingredients. The pumps 12 are preferably of thepositive-displacement type such as piston, peristaltic, gear ordiaphragm pumps. To vary their pumping rate they are preferably drivenwith an electronic speed controller that optionally includes amicroprocessor to calculate the pumping speed and control its variationwith time.

An alternative to the variable rate pumps 12 would be the provision ofvariable valves in pressurised fluid supply lines. The fluid flow ratesdownstream of the valves would depend on the upstream pressures, thevalve openings and the back pressure from downstream fluid controldevices. Flow rate monitors could be incorporated to allow feedbackcontrol over the fluid flows.

Other sensing devices may be provided downstream of the mixing device13, to provide indications of flow rate, pH, conductivity and/or otherdesired parameters. Measurements obtained from such sensors may then beused to adjust fluid flow rates to obtain a desired mix. Such adjustmentmay be real-time, using measurements taken during processing to correctthe fluid mixing in a dynamic way. Alternatively, the measurements maybe used during a separately conducted calibration or characterisationprocess. In this latter case, the performance of the pumps (or otherfluid flow control devices) is characterised by analysing the fluid mixresulting from various predetermined operating rates.

The sensing devices should also be calibrated; this can be donesemi-automatically using standardised solutions in the reagentreservoirs, the standards being passed individually (and unaltered)through the sensors. The sensor outputs may be used to correctsubsequent sensor outputs during an assay, to achieve greater accuracy.Alternatively, calibration may be performed by setting the system tocreate a mix of particular (dynamically measured and real-timecorrected) characteristics. This fluid may be collected and checkedmanually, and the process may be repeated for a variety of mixes and theresults used to correct inaccuracies in the mixing and monitoringsystems.

Fluid connections to the slide cassettes 8, and fluid conduits withinthe cassettes, are arranged to offer similar resistance-to-flow for eachcassette. This means that the fluid flow will divide evenly between thecassettes. Resistance-to-flow can be matched between the cassettes bymatching the length and aperture of conduits to, through and from thecassettes. Where matching is not practical for a part of a conduit (forexample, in the case of a manifold), its internal cross section must bemade larger and/or its length shorter to ensure its resistance-to-flowdoes not affect the division of fluid flow between the cassettes. Thus,the operating rates of pumps such as 12 may be used to control the rateof fluid flow through all of the cassettes.

Clearly apparatus according to the invention may include more than onemixing device 13, and a corresponding number of “sets” of valves andpumps, to allow greater versatility in the number and ratio of fluidswhich can be supplied to the slide cassettes.

FIG. 3 shows in more detail, although still schematically, the fluidcontrol devices in one of the FIG. 2 cassettes 8. Each cassette is anassay device constructed in accordance with the first aspect of theinvention; it contains all the fluid control devices necessary forcarrying out a chemical assay on a test substrate held within it, inparticular devices for storing small volumes of sample fluids andinjecting them into a sample chamber containing the test substrate, foragitating fluids in the sample chamber and for removing fluids from it.

FIG. 3 shows a test substrate 16 (in this case, a microscope slidecarrying a protein array as described above) held in the cassette. Theslide is removable from the cassette and replaceable so as to carry outfurther assays using the same cassette. A cover 17 provides an enclosedsample chamber over the active surface of the substrate 16.

The fluid control devices, all contained within the cassette asdescribed below in connection with FIGS. 9, 12 and 13, include a fluidinlet valve 18 and outlet valve 19 which connect the cassette to thefluid supply lines 10 of the FIG. 2 apparatus and to the outlet conduit15 respectively. They also include sample loading means 20 and 21, eachof which comprises a fluid loading device as described below inconnection with FIG. 20 and a fluid storage device as described inconnection with FIG. 7. These allow storage of sample fluids and theirintroduction into the sample chamber. The cassette also includesagitation devices 22 and 23 which operate in tandem to move fluids backand forth through the sample chamber.

Each of the fluid control devices 18 to 23 is individually supplied withcontrol fluid (eg, compressed air) from the supply line 4 (see FIG. 2).The control fluid supply may be independently controlled for each of thefluid control devices, using conventional fluid flow controls (notshown).

The fluid control devices 18 to 23 are all constructed using twoadjacent plates with a flexible diaphragm sandwiched between them. Theconstruction of the inlet valve 18, for instance, is shown schematicallyin FIG. 4 a (“plan” view) and FIG. 4 b (cross section). It is formed inupper and lower plates 24 and 25 respectively, with a flexible,impervious diaphragm (membrane) 26 clamped between them. The upper plate24 carries a control port 27, to which a control fluid (typicallycompressed air, at for example 300 kPa) can be selectively supplied, anda control chamber 28. Lower plate 25 contains a fluid outlet port 29,leading to the sample chamber of the cassette, and a fluid inlet conduit30. Fluid from the supply lines 10 is supplied to the conduit 30 at amoderate differential pressure (typically 20 kPa) relative to that atthe outlet port 29. In the absence of pressure, the control port 27 maybe vented or, optionally, a negative pressure (relative to that atoutlet port 29) may be applied.

Pressure at control port 27 forces the diaphragm 26 against the upperface of plate 25 over the area of the control chamber 28. The diaphragmthus seals the end of conduit 30, preventing fluid flow through thedevice. If port 27 is vented (or a negative pressure applied to it) thenthe diaphragm is no longer clamped to the plate 25 and may move away,aided if necessary by the pressure of incoming fluid in conduit 30. Thisincoming fluid may then flow to the outlet port 29, and thence into thesample chamber of the cassette. In the case where the valve is “open”when its control port 27 is vented, the valve constitutes a restrictionto fluid flow, which may be overcome by fluid pressure in the inletconduit 30. In contrast, an arrangement in which a vacuum is applied tothe control port in order to open the valve can present less of arestriction to fluid flow.

The supply of all necessary fluids to the sample chamber may becontrolled using valve(s) of the FIG. 4 type.

The diaphragm 26 may be made from any of a variety of materials or evena combination. If the diaphragm material is thin and/or soft then littlepressure is required to force fluid through the valve (supposing port 27to be vented). In contrast, if it is thicker, harder and compressed bythe clamping of the plates then a substantial pressure differential isrequired between ports 27 and 29 to overcome the natural sealing forceprovided by the diaphragm. In this latter case, fluid flow through thevalve may be controlled by varying the pressure of the fluid feedthrough inlet conduit 30, the pressure at the control port 27 remainingconstant (eg, vented).

Clearly the diaphragm should be inert with respect to the fluids passingthrough the valve. This may be achieved either by fabricating thediaphragm from a suitably inert material or by using a laminatedstructure in which a material chosen for its mechanical properties isfaced by a preferably thin layer of a different, inert material. Typicaldiaphragm materials would be silicone rubber sheet (of hardness 45 Shore“A”) faced by a polypropylene sheet. It is not necessary for thecomponents of the laminate to be joined mechanically for the valve tofunction but it may aid assembly.

The operating pressure of the valve is dependent also on its dimensions.A smaller diameter for the control chamber 28 and/or a thicker diaphragm(with a correspondingly increased clamping force) would lead to a higheroperating pressure. Typical dimensions for the FIG. 4 valve would be acontrol chamber diameter of between 3 and 6 mm, preferably between 4 and5.5 mm, a control chamber depth of between 0.2 and 2 mm, preferablybetween 0.5 and 1.5 mm, such as 1 mm, and a diaphragm thickness ofbetween 0.2 and 1.5 mm, preferably between 0.7 and 1.3 mm, such as 1 mm,for a rubber diaphragm of hardness 40-60 Shore A (preferably a siliconerubber of hardness 45 Shore A). Control port pressures in the region of70-300 kPa, preferably 100-200 kPa, such as 150 kPa, would be requiredto operate such a valve.

To prevent undesirable fluid leakage at the edges of a fluid-containingconduit or chamber such as the control chamber 28, outlet port 29 orinlet conduit 30, particularly when the fluid is at a relatively highpressure, the modification illustrated in FIG. 23 may be utilised. FIG.23 a shows schematically part of a fluid control device similar to theFIG. 4 valve, in which a flexible diaphragm 158 is clamped betweenessentially flat upper and lower plates 159 and 160 respectively,spanning the open end of a fluid conduit or chamber 161. The risk offluid leakage from the chamber 161 depends on the pressure applied tothe diaphragm 158 immediately adjacent the chamber edges.

To reduce this risk, as shown in the exploded sectional view of FIG. 23b, one of the internal plate surfaces may carry raised portions such asthe ridges 162 adjacent or close to the chamber perimeter; these serveto concentrate the clamping force applied to the diaphragm 158 aroundthe chamber edges, as shown in FIG. 23 c. As a result, effectiveleak-proof sealing can be achieved by applying a lower clamping force.

Although FIGS. 23 b and c show the provision of raised surface elementsin the lower plate 160, such elements could instead or in addition beprovided in the upper plate 159 and/or in the diaphragm itself, in theregion immediately surrounding the fluid conduit or chamber. Other formsof surface profiling, which achieve the same force-concentrating effectas the ridges 162, may be used.

The FIG. 23 modification may be used in any part of a fluid controldevice according to the invention where sealing of a flexible diaphragmis required around a fluid-carrying channel or cavity. In particular,the modification may be used in devices such as the valves, fluidstorage devices, fluid agitation devices and fluid loading devicesdescribed below in connection with FIGS. 5 to 8, 10, 12, 13, 17 to 20,24 and 25, and/or to enhance sealing around sample chambers.

An alternative inlet/outlet valve, useable as valve 18 or 19 in FIG. 3,is shown schematically in FIGS. 5 a (“plan” view) and 5 b (crosssection). Parts corresponding to those of the FIG. 4 valve arecorrespondingly numbered, and similar comments apply as to theirconstruction and operation.

In the FIG. 5 valve, fluid inlet/outlet conduits 31 and 32 are formed asblind-ended channels in the lower plate 25; both may function as eitherinlet or outlet conduits in use, or the valve may be bi-directional.Control is again effected through control port 27, as described inconnection with FIG. 4.

In a valve such as that of FIG. 4 or 5, it is preferred that the fluidinlet and outlet ports (the ends of the fluid inlet and outlet conduits30 and 29 in FIG. 4) be located as close as possible to the centrallongitudinal axis of the control chamber (28 in FIG. 4), since efficientvalve operation, and in particular effective sealing between thediaphragm and the fluid ports, is less easily achieved towards theperiphery of the relatively large diameter control chamber. For example,one of the ports may be positioned on or very close to the central axisof the control chamber. The other port may also be closer to the centralaxis than to the perimeter of the control chamber, or at least as close.

For example, the valve control chamber may be generally cylindrical inshape and have a cross sectional diameter of about 5.5 mm. The fluidinlet, outlet and control ports might each typically have a diameter ofbetween 0.5 and 2.0 mm, such as about 1.0 mm. In more general terms, thecross sectional diameters of the fluid ports or conduits are typicallybetween 1/20 and ⅕ of that of the control chamber, and the smallestdistance between the perimeters of the inlet and outlet ports (typicallymeasured along a diameter of the control chamber) is then preferablybetween 1/10 and ½ times the control port diameter.

In the FIG. 4 valve, one of the inlet/outlet ports is positionedcoaxially with the control chamber. The central longitudinal axis of thesecond port might then be spaced by 2 mm from that of the controlchamber (ie, the smallest distance between the perimeters of the firstand second ports would be 1 mm).

A particularly preferred alternative form of the FIG. 4 or 5 valve isconstructed as shown in stylised plan view in FIG. 26 a and in crosssection in FIG. 26 b. Reference numeral 177 represents the controlchamber, 178 the control port and 179 and 180 the fluid inlet and outletports. 181 and 182 are upper and lower plates respectively, betweenwhich a flexible diaphragm 183 is clamped. All three fluid portsapproach the control chamber with their central longitudinal axessubstantially parallel to that of the control chamber. Here, the centrallongitudinal axis of each of the inlet and outlet ports is suitablylocated within a distance of ⅛ to ¼ times x from the centrallongitudinal axis of the control chamber, where x is the cross sectionaldiameter of the control chamber.

Further alternative valve constructions are shown in FIGS. 17 to 19.These valves, the general construction of which is illustrated in FIG.17, are set to be either closed (the FIG. 18 valve) or open (FIG. 19) inthe absence of control fluid pressure.

Referring to the schematic “plan” view of FIG. 17 a and cross section ofFIG. 17 b, a valve is constructed between an upper plate 120 and a lowerplate 121, with a flexible diaphragm 122 clamped between them. Providedin the lower plate are a control port 123 and control chamber 124, whichallow pressurised control fluid to displace the diaphragm locallyagainst the opening of a fluid port 125 provided in the upper plate.Fluid may normally flow in either direction between the fluid port 125and an annular groove 126 and channel 127, passing between the upperplate 120 and the diaphragm 122. If however the control port 123 ispressurised, such fluid flow is prevented.

Two alternative forms of such a valve are shown in schematic crosssection in FIGS. 18 and 19; their operation depends on the depth of theupper plate surface in the region 128, around the opening of the fluidport 125 adjacent the diaphragm. If the surface region 128 extends fullyinto the annular groove 126, as in FIG. 18, it distends the diaphragm122. In this case the elasticity of the diaphragm provides a sealingforce to close the fluid port 125, and the valve is “normally closed”.The sealing force can be overcome either by excess fluid pressure (ineither the fluid port 125 or the channel 127), or by application of arelatively low pressure at the control port 123.

A “normally open” valve is shown in FIG. 19. Here, the surface region128 extends only partially into the groove 126 and is therefore clear ofthe diaphragm. The valve is therefore open unless a relatively highpressure is applied at the control port 123.

A “normally closed” valve is generally desirable where it is necessaryto seal against fluid flow in the absence of energisation. A typicalexample might be a valve associated with a fluid storage or loadingdevice, where it is desirable to load the device remotely from the restof the assay apparatus.

A “normally open” valve has a lower resistance to flow at any givencontrol port pressure and might therefore be preferred in locationswhere pressure drop is a potential problem, for instance where fluidsare distributed between several assay devices and variable pressure dropacross the inlet and outlet valves could cause a variable division offlow between the devices.

FIG. 6 shows how the fluid agitation devices 22 and 23 in FIG. 3 may beconstructed in a similar fashion to the valves 18 and 19. The FIG. 6device comprises an upper plate 33, a lower plate 34 and a flexiblediaphragm 35 clamped between them. A control chamber 36, typicallylarger than that of the valve 18, is provided in the lower surface ofthe plate 33. Control port 37 is supplied with control fluid from supplyline 4. Fluid inlet/outlet port 38 communicates with the sample chamberof the cassette.

Using the pressure at port 38 as reference, the application of negativepressure at control port 37 draws the diaphragm 35 away from the lowerplate 34 so that fluid is drawn into the device, from the samplechamber, through port 38 to fill the space between the diaphragm 35 andthe lower plate 34. This situation is illustrated in FIG. 6, the arrowsindicating the directions of fluid flow. Negative differential pressureat control port 37 can be achieved by applying either a negative gaugepressure to the control port or a positive gauge pressure at the port38. Conversely, a positive pressure at control port 37 ejects fluid inthe device back out through port 38.

In this way, fluctuations in applied pressure can be used to move smallamounts of fluid into and out of the sample chamber, thus ensuringcontinuous fluid movement in the region of the test subtrate. Byoperating a pair of such devices in tandem through a sealed volume, backand forth fluid motion can be caused simply by applying positive gaugepressure to the control ports of the two devices alternately.

Generally speaking, gentle rather than vigorous fluid movement will bedesirable throughout the assay device, in particular within the samplechamber. To achieve this, moderate pressures (eg, up to 120 kPa, forinstance about 100 kPa) should ideally be applied to the fluid devicecontrol chambers (such as chamber 36 in the FIG. 6 device), and changesin control fluid pressure should be effected gradually, for instance byincluding a flow restrictor in the control fluid flow. Suitably a periodof between 0.5 and 2.5 seconds, preferably between 1 and 2 seconds,should be allowed for a device such as a valve to be switched betweenstates (eg, between “open” and “closed” or between “on” and “off”).

The sample volume displaced by the FIG. 6 device is dependent on thevolume and cross sectional area of control chamber 36 and the movementof diaphragm 35. These can be set in two ways. If the control chamber isrelatively deep and the diaphragm relatively stiff then the degree ofdiaphragm movement is determined by the applied differential pressure.This may be an advantage in some circumstances where it is desired tochange the displaced volume by remote control; varying the appliedpressure, either manually or automatically, can be used to set thedisplaced volume. In contrast, if the chamber 36 is relatively shallowand the diaphragm more flexible then the diaphragm may be displaced byapplied pressure until it substantially contacts the top face of thechamber. In this case the displaced volume is dependent more on thedimensions of the control chamber and less on the applied differentialpressure. The advantage of this latter arrangement is that apredetermined volume of sample fluid, which does not vary significantlywith applied differential pressure, can be displaced. This could beuseful, for example, where the fluid pressure at port 38 is uncertain.

A device similar in construction to that of FIG. 6 may be used to storea small quantity of fluid (typically a sample fluid) prior to itsintroduction into the sample chamber of the cassette. Another sealingdevice (typically a valve) is required to hold the fluid within thecavity formed between the diaphragm 35 and the lower plate 34. If thissealing device is opened (which may be arranged to occur automaticallyunder the action of excess fluid pressure) then application of pressureat control port 37 will force stored fluid out of the cavity and intothe sample chamber. Such an arrangement is illustrated in FIG. 7.

Two or more fluid control devices such as those of FIGS. 4 to 6 and 17to 20 may be constructed together in a single unit. FIG. 7 illustratesschematically both in “plan” (FIG. 7 a) and in cross section (FIG. 7 b)how this might be achieved. Control port 39, inlet port 40, controlchamber 41 and “intermediate” channel 42 together form a valve. Controlport 43 and storage chamber 44 together function as a fluid storagedevice, communicating with the valve via intermediate channel 42. Fluidcan be trapped in the storage chamber 44 by the action of the valve.Valve sealing may be effected either by pressure applied at port 39 orby the natural elasticity of the diaphragm 45.

To load the FIG. 7 device with for example a sample fluid, the fluid isinjected under pressure through port 40. With sufficient differentialpressure between ports 39 and 40, fluid is pushed from port 40, betweenthe diaphragm 45 and the lower plate 46, into the intermediate channel42. From channel 42 it flows into the storage chamber 44, filling thespace between the diaphragm and the lower plate and distending thediaphragm as it does so.

Once the storage chamber 44 is filled, the fluid is retained by thevalve (either with applied pressure or by elasticity) until pressure isapplied to port 43 in the upper plate 47. This pressurises the fluid toovercome the sealing of the valve (any pressure at port 39 may bereduced or removed) and the fluid exits, via port 40, to the samplechamber. The storage chamber may thereby be either wholly or partiallyevacuated.

Another useful combination of fluid control devices is illustrated inschematic FIGS. 8 a (“plan” view) and 8 b (cross section). Here, a valve(control port 48, control chamber 49 and channels 50 and 51) is combinedwith a fluid agitation device (control port 52, control chamber 53 andsample fluid port 54). An outlet chamber 55 is provided in the lowerplate 56, between the flexible diaphragm 57 and the port 54. In use, thefluid path is from channel 50, through the valve to channel 51 andthrough the agitation device chamber 55 to port 54. Reverse flow is alsopossible. In the absence of pressure at valve control port 48, fluidapplied under pressure to channel 50 displaces the diaphragm 57 in thevalve control chamber 49 to allow flow through to channel 51. Ifpressure is applied to control port 48 then this flow is prevented. Flowthrough channel 51 fills chamber 55 and the fluid can then exit throughport 54 into the sample chamber. Fluid flow through the chamber 55 willsubstantially clear bubbles from it provided its dimensions are not toolarge in comparison to the typical fluid meniscus dimension. Once thechamber 55 is substantially filled with fluid the valve may be closed byapplication of pressure at valve control port 48.

To ensure fluid movement in the sample chamber, pressure, and optionallyvacuum, can be applied cyclically to the agitation device control port52. This makes the diaphragm 57 displace between the chambers 53 and 55,thereby displacing fluid back and forth through port 54.

An advantage of this over the previously described agitation arrangement(FIG. 6) is that the chamber 55 may be thoroughly cleared of bubblesprior to agitation. A similar arrangement may be employed in a fluidstorage device such as that seen in FIG. 7, by the provision of anadditional fluid port to allow the introduction of wash or other fluidsinto the storage chamber. Once filled with such fluid(s), the storagedevice can be purged by operating it as previously described, and thuswashed clean and purged of bubbles prior to its re-use with freshfluids.

A typical assay device (cassette) in accordance with the first aspect ofthe present invention would have two of the FIG. 8 device combinationsconnected to its sample chamber, as seen in FIG. 3. Preferably, thesample fluid ports 54 of the two devices would communicate with oppositeends of the sample chamber so that operation of the agitation devices inopposition would cause fluid displacement over substantially the wholeactive area of the test substrate.

In an arrangement such as that shown in FIG. 8, the valve may be used torelieve excess pressure in the sample chamber, such as might be inducedby raising the temperature in the chamber during a processing operation.This is achieved by applying a predetermined “threshold” pressure to thevalve control chamber 49, causing the valve to open if the fluidpressure in the sample chamber exceeds that threshold.

Generally speaking, for all of the fluid control devices describedabove, very high or very low control fluid pressures are likely to causeundesirably rapid switching between operating positions (eg, betweenclosed and open valve positions). This in turn may lead to sudden fluidmovements which again are undesirable, especially in the sample chamber.Thus, moderate control fluid pressures are ideally used, and changes influid pressures effected as gradually and smoothly as possible.

It will be evident from the above that a complete set of fluid controldevices for each cassette can be constructed simply from two adjacentplates and a flexible diaphragm or membrane between them Each devicetype can be characterised by an arrangement of chambers, conduits andfluid ports provided in the adjacent plate faces.

FIG. 9 illustrates a fluid control unit which can form part of an assaydevice (eg, the FIG. 3 cassette) in accordance with the first aspect ofthe invention. The unit combines two sample fluid storage devices 58 and59, two fluid agitation devices 60 and 61 and two valves 62 and 63 (onefor fluid entry into and one for fluid exit from the cassette). Thestorage devices 58 and 59 are shown with their associated valves, as inFIG. 7—note that the valves in this case are of the “normally closed”type illustrated in FIG. 18; they have no control port and are openedinstead by excess sample fluid pressure. FIG. 9 a is a stylised planview of the fluid control unit and FIG. 9 b a perspective view.

All of the fluid control devices are constructed within upper and lowerplates 64 and 65 respectively, between which is clamped a flexiblediaphragm 66. Holes in the upper face of plate 64 provide control ports67 to 72, as well as reagent fluid ports 73 and 74. Sample fluid ports,which will communicate with the sample chamber in use, are provided asholes (eg, 58 a and 59 a) in the lower face of plate 65.

FIG. 10 is a schematic section through the valve labelled 63 in FIG. 9a. Hole 74 is a fluid inlet port, 75 a sample fluid port intended tocommunicate with a sample chamber and 72 the control port. When thevalve is activated, fluid is allowed to flow through port 75, betweendiaphragm 66 and lower plate 65, through intermediate channel 76 andthrough port 74 (which passes through a hole in the diaphragm). Fluidmay flow either from 74 to 75 or vice versa.

An important feature of the slide cassette (assay device) describedabove is the means to enclose a small volume of fluid in contact withthe active surface of a test substrate. During a typical chemical assay,liquid needs to be passed over the substrate surface to wash it and toapply reagents. However a critical requirement is to be able to leave asmall quantity of a scarce or valuable liquid (such as a biologicalsample) in contact with the substrate for extended periods, typicallymany hours. Ideally the liquid should be spread in a thin layer,covering as much as possible of the active surface of the substrate. Alarger area of coverage allows a larger array of probe species to beincluded. While the is liquid remains in contact with the substrate, itis essential to prevent its depletion by leakage, evaporation orabsorption. These requirements can be met, according to the presentinvention, in a compact assay device of relatively simple construction.

A preferred way in which to achieve efficient sealing of the samplechamber, in for example the cassettes 8 of FIG. 2, is illustratedschematically in FIGS. 11 a, which is a stylised plan view of a testsubstrate and its support, and 11 b, which is a cross section of thesame.

In the FIG. 11 arrangement, the test substrate 77 is clamped against aplate or block 78 with a gasket 79 between them. Typically the testsubstrate is a thin glass plate such as a microscope slide. A clampingplate 80 presses on the back of the slide, which is achieved using anysuitable clamping means. The clamping means preferably includes somemechanical means, such as a spring, to accommodate small differences orchanges in the overall thickness of the assembly. It is necessary thatsufficient clamping force be applied under all combinations of componenttolerance and thermal expansion/contraction or compression set (eg, ofthe gaskets and diaphragms in the device).

Aperture 81 in the gasket 79 defines a sample chamber (82) adjacent theactive surface 83 of the test substrate. It is in this active area ofthe substrate that an array of probe species will previously have beenplaced. Gasket 79 not only seals the substrate against the plate 78 butalso sets the depth of the sample chamber 82. Material for the gasket ischosen for impermeability, softness to conform to the mating surfacesand incompressibility to maintain a reliable thickness under clampingpressure. With a typical slide size of approximately 26 mm by 76 mm, asuitable overlap of the slide over the gasket at each edge is around 2mm.

Though as small as possible a fluid depth is desirable to minimise thevolume required to fill the sample chamber 82, irregularities in theslide and plates make it expensive (because of the tighter finishingtolerance required) to maintain a consistent thickness of less than afew tens of microns. Consequently, a typical sample chamber depth wouldbe around 70 microns. At these dimensions, suitable materials for thegasket are low density polyethylene (LDPE), high density polyethylene(HDPE) and polypropylene (PP). These materials are readily available inextruded films of controlled thickness; gaskets may be cut from suchfilms by known techniques such as punch-and-die or laser cutting orusing knife tools.

For optimum sealing, it is preferable to use the softest possiblematerial consistent with the operating temperature range. For instance,for operation between about 5 and 40° C., LDPE is suitable, whereas athigher temperatures HDPE or PP should be used. This is because, of thethree polymers, LDPE has the lowest softening temperature and PP thehighest. If the gasket is made from a polymer that softens at a lowtemperature compared to the operating temperature of the cassette thenthe clamping pressure may make the gasket extrude from between the testsubstrate 77 and the supporting plate 78.

Since polymers with higher softening temperatures are normally harder atany given temperature there is a potential sealing problem when workingover a wide temperature range. A polymer suitable for withstanding themaximum operating temperature may be too hard at the minimum temperatureand so not conform to the mating faces, allowing leakage. A solution tothis is to use a multilayer material, for instance having a core of aharder polymer, capable of withstanding the higher temperatures, and athinner, softer polymer laminated onto its two faces. The softermaterial effects the sealing but does not suffer excessive extrusionbecause it is such a thin layer. Its viscosity, even at the maximumoperating temperature, does not allow it to extrude from between theplates even over extended processing periods.

In use, fluids may be passed to and taken from the sample chamber 82through small diameter conduits (not shown) provided in plate 78. Theinternal surfaces of these conduits, and the upper surface of plate 78,are both in contact with sample fluid for extended periods and so mustbe made from inert materials. The surfaces must also be, and remain,flat and smooth both for efficient sealing and to prevent unwantedbinding between the surfaces and species in the sample fluid. Suitablematerials include stainless steel (preferably grade 316) and polymerssuch as polyetheretherketone (PEEK), polyoxymethylene (POM—otherwiseknown as acetal), polytetrafluoroethylene (PTFE) or polypropylene (PP).

A significant problem with certain polymers is their absorption ofliquid solvents, particularly water. Assay samples are often formulatedin aqueous solution so it is essential to minimise water absorption intothe components enclosing a sample during potentially extended processingperiods. Absorption into the gasket 79 is not normally a significantproblem because little solvent can be absorbed into the small amount ofmaterial involved and because the area exposed to the sample fluid isvery small. Absorption into plate 78 and into the test substrate itselfcould be much more critical. The test substrate normally poses noproblem since glass, which is the typical substrate material, showsminimal absorption. Absorption into plate 78, however, may prevent useof polymers. A preferred material for the plate is therefore stainlesssteel, which has very low water absorptivity and may be finished to ahigh degree of flatness and polish (by chemical polishing, abrasivepolishing or diamond facing for instance).

The sample chamber construction illustrated in FIG. 11 is compatiblewith the fluid control unit of FIG. 9 to create a complete assay devicein accordance with the first aspect of the invention. Such a device isshown in schematic cross section in FIG. 12. It consists of threeparallel plates, an upper 84, an intermediate 85 (corresponding to thelower plate 78 in FIG. 11 and also to the lower plate 65 in FIG. 9) anda lower “fluidic” plate 86 corresponding to the upper plate 64 in FIG.9. A test substrate 87 is clamped between plates 84 and 85, with agasket 88 (analogous to gasket 79 in FIG. 11) which serves to define anenclosed sample chamber 89. A flexible diaphragm 90 is clamped betweenplates 85 and 86.

The fluid controls for the FIG. 12 device are made up of chambers andchannels (as described in connection with FIGS. 4 to 10 and 17 to 20)defined in the adjacent faces of intermediate plate 85 and fluidic plate86, together with fluid conduits through the two plates and thediaphragm 90. Such chambers, channels and conduits are omitted from FIG.12, for simplicity.

In this case, the assay device might include:

-   -   i) two sample storage and injection devices, as illustrated in        FIG. 7, communicating with the sample chamber 89;    -   ii) two inlet/outlet valves, as illustrated in FIG. 4, 5, 10 or        17 to 19, to control fluid flow from external reservoirs to the        sample chamber or from the sample chamber to waste; and    -   iii) two fluid agitation devices, as illustrated in FIG. 6 or        FIG. 8, to move fluid contained in the sample chamber back and        forth across the active surface of the test substrate 87.

An assay device such as that of FIG. 12 may also incorporate one or morefluid loading devices of the type shown in schematic cross section inFIG. 20. This comprises an open cup-shaped recess 129, of approximatevolume 50-100 μl, provided in the outer surface of upper plate 130. The“cup” 129 has first and second outlets 131 and 132 respectively.

Outlet 131 leads to a valve which is constituted by a control chamber133, a control port 134, an intermediate chamber 135 and a fluid outletport 136. The valve is operated by the supply of control fluid to thecontrol chamber 133 which causes displacement of the diaphragm 137 andthereby controls fluid flow either into or out of the cup 129. In thiscase, fluid outlet port 136 leads to a sample chamber.

The second outlet, 132, from the cup leads to waste (outlet port 138).

The FIG. 20 device is provided in a three-plate construction whichincludes not only the upper plate 130 but also a pair of lower plates139 and 140. A second flexible diaphragm 141 is located between the twolower plates. Such a construction has the advantages described below inconnection with FIG. 25.

Fluid may be loaded into the cup 129 either directly or, as shown inFIG. 21, by insertion of a pipette tip 142 into the first outlet 131.The mouth of outlet 131 is specially adapted to accommodate a standardpipette tip.

The valve associated with the FIG. 20 loading device is analogous inoperation to those described in connection with FIGS. 4, 5, 10 and 17 to19. To open the valve, a low pressure is applied to its control chamber133, This causes a local downwards displacement of the diaphragm 137,which allows fluid flow either to or from the cup 129. The applicationof a higher pressure to the control chamber 133 seals the diaphragm 137against the upper plate 130, preventing fluid flow into or out of thecup.

The FIG. 20 device may be used to load fluids into other parts (inparticular the sample chamber and/or fluid storage devices) of the assaydevice, and to evacuate fluids from other parts such as the samplechamber. It may also be used in the washing of the sample chamber andother apparatus parts.

A typical sample loading operation would involve dispensing sampleliquid either directly into the cup 129 or via a pipette as shown inFIG. 21. If the cup has previously been washed (as described below),then a small quantity of wash liquid will remain in it and this ensuresthat the sample can be delivered to the valve inlet under a liquidsurface so that no bubbles can be trapped.

Sample aspiration may then be achieved by opening the control valve soas to draw the sample liquid through into the sample chamber by vacuumapplied downstream of the valve. This could be done for instance byoperating a fluid storage device (of the type described in connectionwith FIG. 7) to suck the liquid in.

Just as liquid can be drawn in from the cup 129 by vacuum, similarly itmay be expelled to the cup (or to a pipette tip inserted into the cupoutlet 131) by appropriately applied pressure.

A typical washing operation may be achieved using the FIG. 20 device byintroducing a wash liquid to the valve (for instance via the port 136)under slight pressure. Vacuum is then applied to the control chamber133, allowing the wash liquid to feed into the cup 129. Vacuum appliedto the waste port 138 removes excess liquid from the cup, takingcontaminants away with it. Any bubbles trapped in the valve, inparticular in its inlet conduit, are also purged in this process. Oncethe supply of wash liquid is stopped, fluid in the cup drains down toapproximately the level of the waste outlet 132.

FIG. 22 shows schematically how a group of fluid loading devices, of thetype shown in FIG. 20, may be used together in an assay device accordingto the invention. Here three loading devices, 143 to 145, areillustrated. Each has an associated valve (146 to 148 respectively).Items 149 and 150 are fluid storage devices, each associated with asample chamber in which an assay is to be conducted. Items 151 to 155are further fluid flow control valves. Conduit 156 is connected to asource of wash fluid, and conduit 157 leads to waste.

All three loading devices are in fluid communication with storage device149, and loading device 145 is additionally in fluid communication withstorage device 150. All three loading devices may be used to deliverfluids to storage device 149 and to evacuate fluids from it (forinstance, previously stored or assayed sample fluids, or wash liquids).All three loading devices may be washed with fluid supplied via thestorage device 149. In addition, device 145 may deliver fluids to orreceive fluids from the storage device 150. Fluids may also betransferred between all three loading devices via the storage device149.

The capacities of the loading device “cups” may be sufficiently greatthat they may be only partially filled or emptied in any given fluid“transaction”. Thus, for example, device 145 may be used to dispensealiquots of fluid to both the storage devices 149 and 150.

A major advantage of the FIG. 22 arrangement is the flexibility itoffers in terms of fluid movements. It makes possible not only thestorage of small quantities of a number of different fluid samples, butalso the drawing of more than one fluid sample from each loading deviceand the supply of fluid to any given sample chamber or storage devicevia more than one loading device. Operation of the loading devices canmoreover be automated, conveniently via operation of other fluid controldevices in the surrounding apparatus, with which the fluid loadingdevices are operationally compatible.

The fluid loading devices also offer the ability to purge air bubblestrapped in the device, whilst their compatibility with conventionallaboratory pipettes makes them straightforward to use.

Ideally, fluid loading device(s) are located in the assay device of theinvention in reasonably close proximity to the sample chamber. There mayhowever be cases in which one or more fluid loading devices are providedat a different location, for instance as part of an assay station atwhich one or more assay devices are to be processed.

Fluid connections are made to the FIG. 12 assay device through holes(not shown) in the lower face of fluidic plate 86, for entry and exit ofsample and reagent fluids and also of control fluid for the valves andother devices. These connections may be made by individual tubes.However, in a system which has several such assay devices or “cassettes”at an assay station, there will be many such connections and high costmay result. An alternative connection method is to combine the fluidicplates 86 of several cassettes into a single plate. In the lower face ofthis plate are fabricated channels to distribute and collect the variousfluids to and from the cassettes. At least some of the fluid controldevices and fluid distribution conduits may therefore be common to morethan one assay device.

Such an arrangement is shown in stylised “plan” view and in crosssection in FIGS. 13 a and 13 b respectively, FIG. 13 b being a sectionalong the line B-B in FIG. 13 a. Here, fluidic plate 91 is common toseven assay cassettes 92 (in general, there may be any desired number ofcassette locations in an assay station such as that of FIG. 13). Eachcassette comprises a diaphragm 93, an intermediate plate 94, a gasket 95and an upper clamp plate 96. A test substrate 97 is shown clamped insidethe cassette seen in FIG. 13 b, in an arrangement similar to that ofFIG. 12.

The lower face of the fluidic plate 91 provides a number of channels 98which run beneath all of the cassette locations. The channels 98 areclosed at either end except for fluid connection ports 99 through theupper face of the plate. Though they may be fabricated as closed tubeswithin the body of plate 91 (eg, by drilling or extrusion), it is moreeconomic to fabricate them in the plate face and then close them offwith a sealing gasket 100 and gasket plate 101, as shown in FIG. 13 b.The gasket plate 101 may be clamped to fluidic plate 91 by anyconvenient method.

Holes through the fluidic plate 91 in selected positions allowcommunication between the channels 98 and the fluid control devices inthe upper face of the plate and/or the intermediate plate 94, for eachcassette position. Using the fluid ports 99, liquids and gases may thusbe supplied to the cassettes 92 via the channels 98. Any of the channels98 may connect either to a single fluid control device of a cassette orto a particular type of device across all the cassettes. A preferred setof channel functions is listed below.

-   -   (i) liquid (typically wash and/or reagent liquid) source to all        cassettes    -   (ii) to (viii) control fluid source for input valves in        cassettes 1 to 7 respectively    -   (ix) control fluid source for agitation device A (all cassettes)    -   (x) control fluid source for sample A storage/injection device        (all cassettes)    -   (xi) control fluid source for sample B storage/injection device        (all cassettes)    -   (xii) control fluid source for agitation device B (all        cassettes)    -   (xiii)-(xix) control fluid source for output valves in cassettes        1 to 7 respectively    -   (xx) liquid outlet from all cassettes.

With this combination of fluid control devices it is possible to blankoff individual cassettes so that the assay station may be processed withfewer than seven test substrates in place. Otherwise control is commonto all cassettes so that they run synchronously.

A “sub-assembly” such as that of FIG. 13 includes all of the fluidcontrol devices necessary for each cassette. The sub-assembly can formpart of an assay station as shown in FIG. 1, the remaining controlelements including the pumps and valves shown in FIG. 2. Preferably, theFIG. 13 sub-assembly is built as a module that can be easily and quicklyremoved from a station of which it forms a part. Fluid and electricalconnections to the sub-assembly can be made via ganged connectors tofacilitate this.

In fluid control devices of the types described above, and in assembliesor sub-assemblies incorporating such devices, it may be necessary for afluid conduit or chamber provided in one plate to overlay, at leastpartly, another conduit or chamber provided in an adjacent plate. Such asituation is illustrated in FIG. 24 a, which is a section through partof a fluid control device formed between upper and lower plates 163 and164 respectively, in which fluid ports 165 and 166 and their commonfluid conduit 167 are separated by the flexible diaphragm 168 from fluidconduit 169 in the lower plate. The arrangement is shown in stylised“plan” view in FIG. 24 b, the conduits within the structure beingillustrated by dashed lines. FIG. 24 c is a section corresponding toFIG. 24 a, but illustrating how, when the fluid pressure in conduit 169is higher than that in conduit 167, displacement of the diaphragm 168may be sufficient to allow fluid leakage from conduit 169 along theinterface between the diaphragm 168 and the lower plate 164.

This problem may be overcome or at least mitigated by providing an“inner” plate between the two plates in which the relevant overlappingconduits/chambers are defined. Such an arrangement is illustrated inFIG. 25, in which overlapping fluid conduits 170 and 171, provided inupper plate 172 and lower plate 173 respectively, are separated by aplate 174 sandwiched between two flexible diaphragms 175 and 176. Allthree plates are made from a suitable rigid material such as stainlesssteel, a ceramic material or a rigid plastics material. The inner plate174 transmits clamping forces over the whole mating surface of each ofthe upper and lower plates, avoiding unsupported regions in the flexiblediaphragms which might otherwise-allow fluid “tunnelling” between thediaphragms and adjacent plates.

Any of the fluid control devices of the invention, including thosedescribed above and in particular the FIG. 26 valve, may be constructedusing an arrangement of the form shown in FIG. 25.

A further optional feature of apparatus in accordance with the inventionis a device for monitoring the fluid flow rate through one or more ofthe apparatus parts, in particular through assay devices. This isdesirable firstly in order that malfunctions may be detected andsecondly so that pumping rates may be adjusted to achieve a desired flowrate.

A flow rate monitoring device in accordance with the third aspect of thepresent invention, for use for example in the apparatus of FIG. 1, isshown in schematic cross section in FIG. 14. It is provided in a fluidicplate 102, which may simply be an extension of the fluidic plate, suchas 91 in FIG. 13, of an assay “cassette”. Two cavities, 103 and 104, aremachined into the fluidic plate. They are closed by clamping a printedcircuit board (PCB) 105 to the upper face of plate 102 with a gasket 106between. The cavities communicate with each other via a labyrinth 107 offluid conduits provided in the body of plate 102 (this labyrinth mayhave any desired geometry). Fluid from an assay cassette may enter thedevice at port 108 and exit, typically to waste, at port 109. Port 110allows injection of a gas, the purpose of which is described below. Thearrows indicate the directions of fluid flow in use.

Fluid (typically liquid) filling the cavities 103 and 104 comes intocontact with the face of the PCB 105. Its presence can be detected ineither cavity by detecting a change of conductance or capacitancebetween conductor traces provided on the lower face of the PCB; theelectronics to do this may be provided either on the PCB itself orremotely. This provides a digital indication of the presence or absenceof liquid in the cavities.

To measure flow rate, a small bubble of gas is injected, via port 110,into the liquid flowing through the FIG. 14 device. The dimensions ofthe fluid conduits and cavities in the device are such that the bubblefills the cross section and propagates along with the liquid flow. As itpasses the PCB sensors associated with cavities 103 and 104, acontrolling computer detects this and measures the time taken for thebubble to pass through the labyrinth 107. From this, an approximateliquid flow rate can be calculated.

An additional benefit of the FIG. 14 device is that it can also checkfor the presence of bubbles during “normal” liquid flow, for instance asa quality check during purging.

If the speed of gas injection at port 110 can be held sufficientlyuniform, it may be possible to achieve sufficient accuracy with a singlecavity and PCB sensor, the interval between gas injection and sensingbeing the measured parameter.

Clearly, the PCB must be compatible with the fluids present; this may beachieved for instance by constructing the PCB from gold-plated tracks onan epoxy substrate.

Optical or other alternative detection means may be used instead of thePCB to detect the presence or absence of liquid in the cavities.

The FIG. 14 device may be inserted in any desired liquid flow. Forinstance, if the flow rate through each of the cassettes needs to bemeasured independently then one device per cassette is required.Preferably the device is positioned downstream of the cassette, toprevent the injected gas bubbles affecting assays being conducted in thecassette.

The gas injection port could conveniently be common to a number ofcassettes. To prevent liquid flowing along this common connection, whichwould affect the gas injection, a valve of the type shown in FIG. 5could be inserted into the gas supply line. This valve could be operatedeither through its control port 27 or simply by the pressure of the gasfeed overcoming the natural sealing of the diaphragm 26.

It will be evident that both the flow rate monitoring device and anyassociated valve(s) can be fabricated as features in the plates 91, 94and 101 of a cassette (see FIG. 13), making use of the diaphragms 93 and100 between them. This minimises the overall cost of the cassette as thecomponents of all the fluid control devices can be fabricated at thesame time.

During a chemical or biochemical assay it is usually necessary tocontrol the temperature of the test substrate and the fluids in contactwith it. This may involve heating to temperatures above ambient and/orcooling below ambient, often “cycling” between different operatingtemperatures at different times during the processing.

Using apparatus in accordance with the present invention, temperaturecontrol may be effected in a variety of known ways such as by:

-   -   i) passing, heated or cooled air over the whole assembly;    -   ii) allowing heated or cooled liquid to flow against or through        any convenient part of the assembly, provided there is good        thermal conductivity between that part and the remainder;    -   iii) electrical resistance heating of such a part; and/or    -   iv) Peltier heating or cooling of such a part.

A preferred temperature control means is illustrated in schematic crosssection in FIG. 15, in combination with a cassette of the type shown inFIG. 13 (like parts are labelled with the same numerals). In this case,the lowermost plate 111 is an extruded (for instance, aluminum) heatsink of the type conventionally used in electronic systems, having anumber of cooling “fins” 111 a. In this case the heat sink can alsoitself be heated, by means of electrical heaters 112 connected to it.Applying power to these heaters raises the temperature of the heat sink111 and, by conductivity, of the associated cassette. The temperaturemay be monitored by any convenient means, such as platinum resistance orthermocouple sensors. Using an automatic temperature controller, thetemperature of the assembly may be stabilised at any desired level aboveambient.

An enclosure 113 fits around the whole assembly to minimise convectionand other draughts and hence temperature differences within theassembly. However the natural cooling rate of the assembly, in theabsence of heater power, is then reduced, which in turn could slow downits overall operating rate and even be deleterious to the test processitself. Faster cooling may therefore be achieved using a flow of airforced through the heat sink 111 by an electrically driven fan (notshown). Such a fan could also be under the control of the automatictemperature controller system so as to be activated automatically whencooling is required. If cooling below the ambient temperature isrequired then air drawn in by the fan must be precooled using any of avariety of known techniques.

Apparatus in accordance with the fourth aspect of the present inventionmay include any desired number of assay stations, as shown in FIG. 1,each station having a plurality of cassette locations. The apparatusdescribed above, in connection with FIGS. 1 to 15 and 17 to 25, allowsprocessing with any combination of cassettes and assay stations active.

As a result of common fluid connections, active cassette positionswithin a station can be partly interdependent. Each station, however,can operate entirely independently. A controlling computer or othersequencer may be used to operate the valves, pumps, heaters and otherdevices necessary to execute a pre-programmed processing sequence at oneor more stations.

The following describes how the apparatus might typically be operated,by reference to one assay station. Other stations in the apparatus maybe operated, synchronously or asynchronously, in a similar way.

Preparation

Before a processing run may be performed, all washes, reagents etc. mustbe in the relevant reservoirs. It is typically a manual task to checkand fill reservoirs. Waste bottles must also have sufficient remainingcapacity. The apparatus may be connected to “main-line” services such aspurified water, gas and waste in some circumstances. Automatic checkingof reservoir levels may be included by any of a variety of knowntechniques (eg, weight sensor or weight balance).

Station Configuration and Sample Storage

If fewer test substrates are to be processed than the maximum stationcapacity, unused cassette locations must be made inactive. Depending onthe exact configuration of the fluid control devices and the stationassembly, this may require no further action. In the preferredarrangement, however, it is necessary to “blank-off” unused cassettepositions to prevent leakage of control gas or other fluids from exposedports. This is achieved as shown schematically in FIG. 16.

FIG. 16 a is a side view and FIG. 16 b a stylised plan view of a“blanked-off” cassette from the apparatus described above. As in FIG.15, item 111 is a plate which functions both as support and also as aheat sink, item 91 is a fluidic plate carrying fluid control devices andfluid channels and item 100 is the diaphragm between these two plates. Abacking plate 114 and plain sealing gasket 115 are clamped in place ontop of fluidic plate 91, by means of a spring clamp 116 and toggle clamp117.

Ideally, the apparatus is arranged so that even in the “blanked-off”cassettes, the fluid control devices are periodically (and preferablyautomatically) operated, ie, fluid is passed through them, to prevent orat least reduce adhesion between the flexible diaphragms and theadjacent plates.

For each “active” cassette, a diaphragm 93 and intermediate plate 94(see FIG. 13) are used in place of the backing plate 114 and gasket 115,secured using similar spring and toggle clamps. With these in place, oneor more small quantities of sample fluid or other reagent may bepre-loaded into one or more of the storage devices of the cassette,ideally via fluid loading devices, using for instance a pipette orsyringe. (The syringe is ideally fitted with a hollow tip suitable forsealing into the inlet port of the relevant fluid control device.)

Fitting Test Substrates

Once the sample and other necessary fluids have been pre-loaded, thegasket 95, test substrate 97 and clamp plate 96 (FIGS. 13 and 15) may befitted to each active cassette. Again these are conveniently held inplace by spring clamps, as described in connection with FIG. 16. Thetest substrate carries at least one “probe” species which will react(preferably selectively) with a target species contained in or thoughtto be contained in the sample under test. A typical test substrate for abiochemical assay is a glass microscope slide coated with streptavidin,with one or more (preferably an array of) biotin-tagged probes, such asnucleotide sequences, antigens or antibodies, immobilised on it. The useof avidin-biotin binding to immobilise biological reagents on asubstrate is entirely conventional.

Fitting the Station Sub-Assembly to the Assay Station

Once a station sub-assembly of the type shown in FIG. 13 (containing aplurality of cassettes) has been prepared with all of the required testsubstrates, sample fluids and sealing plates for unused positions, it isfitted into one of the assay stations 3 of the FIG. 1 apparatus. Indoing so, fluid and electrical connections are made between the fluidand thermal control devices of the station and those of thesub-assembly.

Automated Processing

The apparatus may then be used to carry out an almost fully automatedchemical assay, typically under the control of a pre-programmedmicrocomputer or other process control means. This is set up to operatethe pumps, valves, thermal controller and other requisite devices in apre-programmed sequence. A typical such sequence involves:

-   -   i) purging the system—in turn, open each valve between        reservoirs and each pump. The pump is run with the manifold        bypass valve 14 (FIG. 2) open.

Liquid runs to waste, clearing all fluid conduits and manifolds as itdoes.

-   -   ii) washing the substrates—selected reservoirs, containing for        instance concentrated wash liquid and distilled water, are        connected to the pump(s) by opening the corresponding valve(s).        The pump(s) are run at a rate dependent on the required fluid        mixing ratio and flow rate, the latter depending on the number        of active cassettes in the assay station. Inlet and outlet        valves for selected cassettes are opened (by appropriate        operation of the associated control valve applying pressure or        vacuum or vent to the valve control ports). Outflow from the        cassettes goes to waste. Active cassettes may be washed        independently or together by appropriate control of their inlet        and outlet valves.    -   iii) applying reagent(s)—as in step (ii), but using reagent        fluid reservoir(s) as the source(s). Reagent fluids may include        buffers, surfactants, electrolytes, catalysts, reaction        initiators and/or terminators, blocking agents, labelled        reagents and the like.    -   iv) injecting sample—with the cassette inlet valves closed but        their outlet valves open, apply pressure to the corresponding        control ports of the sample storage devices. Liquid stored in        the devices is injected into the sample chambers of the        cassettes.

Concurrently with the other steps, the temperature of the cassetteassemblies may be set to a predetermined value by the thermal controlsystem. This may involve heating or cooling, as previously described.

During the assay, pressure (and optionally vacuum) is appliedalternately to the agitation devices of each pair of devices in eachactive cassette (the inlet and outlet valves being closed). This movesthe liquid in the sample chambers back and forth.

Subsequent assay steps may involve washing, heating and/or cooling,agitating and/or supplying further reagents or samples to the testsubstrates and sample fluids, all as described above.

When the assay is complete, gas or air may be pumped through thecassettes to remove most of the liquid in the sample chambers. The testsubstrates may then be removed from the cassettes and appropriatelyimaged to obtain the desired test results.

In the washing step(s), the apparatus of the invention allows the washsolution concentration to be altered as desired. This in turn makespossible several successive washing steps, typically with increasingdegrees of stringency.

It can be seen from the above that apparatus in accordance with thepresent invention can possess several key advantages, namely:

-   -   i) a small volume of reaction or wash fluid can be enclosed        against the test substrate;    -   ii) multiple test substrates can be simultaneously and        economically processed;    -   iii) small quantities of sample fluid can be pre-loaded and        efficiently stored for each substrate under test;    -   iv) multiple samples can be stored for each test substrate,        allowing multiple probing with for instance several different        antibodies;    -   v) operation can be at least partially, preferably fully,        automated;    -   vi) the sample fluid can be agitated over the active surface of        the test substrate;    -   vii) multiple wash or reagent fluids can be introduced into the        sample chamber;    -   viii) wash and/or reagent fluids can be blended to achieve        desired concentrations or mixes that can be varied continuously        with time;    -   ix) large numbers of samples can be assayed simultaneously, with        independent control of the processing conditions (for example,        the temperature and fluid movement) for each;    -   x) the fluid control devices, such as valves and agitators, are        relatively simple and compact in construction, being        incorporated into the cassettes. This allows the use of large        numbers of reagent and sample fluids without undue size,        complexity and cost in the apparatus as a whole.

1. An assay device for use in carrying out a fluid-phase chemical assay,the device comprising: a sample chamber in which a sample may beretained; a valve comprising a control fluid inlet for controlling aflow of control fluid, a reagent fluid inlet, a reagent fluid outletproximate to said reagent fluid inlet for permitting said reagent fluidto enter said sample chamber from said reagent fluid inlet, and adisplaceable diaphragm for controlling the flow of reagent fluid fromsaid reagent fluid inlet to said fluid outlet; said control fluid inletis placed on one side of said displaceable diaphragm; said reagent fluidinlet and said fluid outlet are placed on a side of said displaceablediaphragm opposite said control fluid inlet; wherein said displaceablediaphragm, in a first position, is in contact with both said reagentfluid inlet and said fluid outlet; wherein said displaceable diaphragm,in a second position, is moved away from both said reagent fluid inletand said fluid outlet; wherein said displaceable diaphragm is moved fromthe first position to the second position, and vice versa, dependingupon the flow of control fluid from said control fluid inlet; twoagitation devices, each having a flexible diaphragm placed between anupper plate and a lower plate; said upper plate includes a fluid inletand said lower plate includes a fluid outlet that is in communicationwith said sample chamber; and wherein pressure at either said fluidinlet or said fluid outlet causes said flexible diaphragm to fluctuateand fluid to move into and out of said sample chamber; a storage chamberfor holding reagent fluid; said reagent fluid outlet is connected tosaid storage chamber; said displaceable diaphragm extends from saidreagent fluid inlet to between said storage chamber and said reagentfluid outlet for permitting reagent fluid to enter and exit said storagechamber; wherein said displaceable diaphragm moves away from saidreagent fluid outlet to allow reagent fluid to enter said storagechamber and said displaceable diaphragm moves toward said reagent fluidoutlet to cause reagent fluid to exit said storage chamber; and said twofluid agitation devices are located at opposite ends of said samplechamber for moving fluid back and forth through said sample chamber. 2.An assay device according to claim 1, further comprising a fluid storagedevice in communication with said sample chamber for storing fluid. 3.An assay device according to claim 2, wherein said fluid storage devicecomprises an inlet port and a storage chamber for holding fluid receivedat said inlet port, and wherein a valve controls movement of fluid intoand out of said storage chamber, being displaceable between a firstposition in which fluid is held in said storage chamber, and a secondposition in which fluid exits said storage chamber.
 4. An assay deviceaccording to claim 3, wherein said valve is displaced by an associatedvalve or by pressure change to said storage chamber.
 5. An assay deviceaccording to claim 3, further comprising an intermediate chamber betweensaid storage and said outlet.
 6. An assay device according to claim 2,wherein said fluid storage device stores fluid prior to its introductioninto or following its evacuation from said sample chamber and furthercomprising a valve for controlling the flow of fluid into and out ofsaid fluid storage device.
 7. An assay device according to claim 1,further comprising a fluid loading device for loading fluid into theassay device.
 8. An assay device according to claim 7, wherein saidfluid loading device comprises a receptacle to accommodate fluid and avalve between said receptacle and said sample chamber.
 9. An assaydevice according to claim 7, wherein said fluid loading device includesa first outlet between said valve and said sample chamber and a secondoutlet through which fluid may be evacuated, said second outlet beingpositioned at a higher fluid level within said receptacle than saidfirst outlet.
 10. An assay device according to claim 1, furthercomprising at least two fluid control devices having fluid ports andchambers of which are defined within a single unit and a diaphragmcommon to said at least two fluid control devices.
 11. An assay deviceaccording to claim 10, wherein said diaphragm common to said at leasttwo fluid control devices is positioned between two adjacent plates, andwherein at least some of the fluid ports and chambers of the fluidcontrol devices are provided at one face of the plates and are at leastpartly defined by a sealing layer positioned adjacent the one face. 12.An assay device according to claim 11, further comprising at least twostacked plates and a diaphragm positioned between said at least twostacked plates.
 13. An assay device according to claim 1, furthercomprising a fluid distribution assembly for introducing fluid into thedevice from external sources and subsequently removing the fluid fromthe device, said fluid distribution assembly including a fluid inletport connected to an external source of fluid, a conduit through whichfluid may pass from the inlet port to said sample chamber, a fluidoutlet port, and a conduit through which fluid may pass from said samplechamber to said fluid outlet port.
 14. An assay device according toclaim 13, further comprising two or more adjacent plates with adiaphragm and a conduit positioned between each pair of adjacent plates.15. An assay device according to claim 1, further comprising a substrateon which a probe specie is placed, wherein the substrate and a fluidcontrol unit define the sample chamber, in fluid communication with thefluid control device, in which a fluid sample may be retained in contactwith the test substrate.
 16. An assay device according to claim 15,wherein the substrate is flat.
 17. An assay device according to claim16, further comprising a microscope slide.
 18. An assay device accordingto claim 1, further comprising multiple sample chambers.
 19. An assaydevice according to claim 18, further comprising one or more assaystations.
 20. Apparatus according to claim 1, further comprising a flowrate monitoring device which includes a primary measuring chamberthrough which a first fluid may flow, a fluid inlet port upstream of theprimary measuring chamber, through which a volume of a second fluid maybe introduced into the first fluid flow, and a primary fluid detectorassociated with the primary measuring chamber for detecting the presenceof the second fluid in the first fluid as they pass through the primarymeasuring chamber.
 21. Apparatus according to claim 20, wherein the flowrate monitoring device additionally comprises a secondary measuringchamber, in fluid communication with the primary measuring chamber, thesecondary measuring chamber having associated with it a secondary fluiddetector for detecting the presence of the second fluid in the first asthey pass through the secondary measuring chamber, and wherein fluidcommunication between the measuring chambers is by means of alabyrinthine fluid conduit.
 22. Apparatus according to claim 1, furthercomprising at least partially automated controls.
 23. Apparatusaccording to claim 1, further comprising a fluid distribution systemwhich includes first and second fluid inlet lines via which first andsecond fluids may be drawn; first and second fluid flow control devices,each allowing a variable fluid flow rate in the first and second fluidinlet lines, respectively; a controller for controlling the flow ratesthrough the first and second fluid flow control devices; and a fluidmixing device downstream of the fluid flow control devices for combiningthe first and second fluids emerging from the flow control devices. 24.Apparatus according to claim 23, wherein the fluid flow control devicesallow the flow rates of the first and second fluids to be variablebetween their minimum and maximum values, allowing for variation of thefirst to second fluid ratio in a mixture emerging from the fluiddistribution system.